Study Of The Effects Of Berberine Hydrochloride On Vascular Smooth Muscle Cells In The Type 2 Diabetes Mellitus

Abstract:Objective:To investigate the effect of different concentrations of berberine hydrochloride on the proliferation,migration,and the concentrations of TGF-β1,IL-6 and TNF-a in vascular smooth muscle cells (VSMCS) of type 2 diabetic rats, to study its mechanism and prevention against Diabetic vasculopathy from cellular even molecular level.Methods:(1)Modeling:30 healthy adult male SD rats were included, which weighed 200±20g, these rats were randomly divided into two groups:control group with 10 rats and model group with other 20 rats after 1 week raised.The rats of control group were fed on standard diet,while the rats of model group were fed on the diet enriched in fat and carbohydrate.After 1 month,the rats of model group were injected intraperitoneally with streptozocin(30mg/kg). The rats whose fasting blood glucose (FBG) more than 7.0 mmol and 2 hours blood glucose(2hBG) more than 11.1 mmol were successful to be the diabetic model. (2) Identification of VSMCs:To culture thoracic aortic medial VSMCs of normal rats and type 2 diabetes mellitus rats with the adherent metllod of tissue explants, observe cellular forms by the inverted microscope,observe the cellular growth curve by counting and identify VSMCs by a-SM actin immunohisto-chemistry. (3)Detection index:The normal and type 2 diabetes mellitus rats'VSMCs suspensions which exponential phase of growth from 4 to 6 were seeded in 96 well plate. The normal rats'VSMCs were normal, type 2 diabetes mellitus rats' VSMCs were separed into model group, positive control group, treatment groups 1 to 5 by randomly.The drugs and final concentration in these groups were serum-free DMEM,50μg/ml captopril,100,50,25,12.5,6.25μg/ml berberine hydrochloride.Interference effects of berberine hydrochloride on proliferation(by Cell Counting Kit-8), migration(by transwell) of VSMCs and the level of TGF-β1,IL-6,TNF-a (by ELISA)was detected.Results:(1)Success of modeling:The weight of model rats was 262.11±18.35g, the weight of normal rats was 350.56±19.74g, it wase showed statistically significant (P<0.01). The FBG of model rats was increased significantly from 5.23±0.74mmol/L to 15.82±2.44 mmol/L(P<0.01). The 2hBG of model rats was increased significantly from7.23±1.31mmol/L to 23.78±2.23 mmol/L(P<0.01). The insulin of model rats was increased significantly from 32.59±3.12μIU/ml to 54.38±4.97μIU/ml (P<0.01).The ISI of model rats was -6.37±0.43,the normal was -4.16±0.39,this conclusion showed statistically significant (P<0.05). (2) Under inverted microscope, singel smooth muscle cell of normal rat was spindle thinly, triangular shape,fan-shaped or irregular shap,and cells grew parallelly to the beam when they were dense,overlapping part of the performance as "peak-valley" growth;VSMCs of diabetes mellitus rats were spindle,fan-shaped or irregular shape,a great number cells growing together,more dense than normal,woven into network of overlapping into the whirlpool-like growth; staining the third to sixth generation of diabetes mellitus rats VSMCs by specific immune cellsa-actin,cytoplasmic staining,positive, and we can see that a large number of brown,the filament fiber with the long axis paralleling to the cells,which is a-smooth muscle actin filament.The growth curve of VSMCs similar to "s" shape, the proliferation was exuberant at first three days,and then started decline after 5 days.The growth rate of diabetes mellitus rats VSMCs was significantly fastest than the normal.(3)Detection index:①The effect of proliferation:The absorbance of normal group,model group,positive control group was 0.307±0.025,0.617±0.033,0.465±0.032 respectively, The absorbance of treatment group from 1 to 5 was 0.352±0.023, 0.399±0.029,0.452±0.032,0.505±0.035,0.531±0.044 respectively.Every concentration of berberine can inhibite the effect of proliferation on diabetes mellitus rats VSMCs, and with the increasing concentration of berberine, inhibition increased. Inhibition of the proliferation on treatment group 1 and treatment group 2 was significantly stronger than the positive control group.①The effect of migration:The amount of treatment groups cells that cutting through the cellulose nitrate membranes after effect by berberine was 19.20±1.15,21.27±1.28,25.33±1.95,30.67±1.72,40.00±1.65 high power field respectively,less than model group which was 48.33±2.38/high power field,it has statistically significant(P<0.01).This showed that every concentration of berberine group can inhibite the migration of diabetes mellitus rats VSMCs.③The effect of the concentration of TGF-β1,IL-6,TNF-α:The concentration of TGF-β1 in medium of model group, positive control group, treatment groups 1, to 5 was 22.54±2.96,18.55±2.85,14.56±1.60,15.67±1.10, 18.18±2.82,19.12±1.92,20.17±1.73ng/ml respectively, significant higher than normal group which was 10.96±1.48 ng/ml (P<0.05). The concentration of IL-6 in medium of model group, positive control group, treatment groups 1 to 5 was 469.47±20.47,401.66±20.01,303.51±15.50,340.56±18.60,382.19±17.99, 421.62±22.37,451.15±23.57 pg/ml respectively,significant higher than normal group which was 257.34±17.0pg/ml (P<0.05).The concentration of TNF-a in medium of model group, positive control group, treatment groups 2 to 5 was 2.15±0.30,1.59±0.22,1.27±0.30,1.51±0.18,1.65±0.33,1.89±0.13ng/ml respectively,significant higher than normal group which was 0.86±0.20ng/ml (P <0.05). Berberine can reduce the concentration of TGF-β1,IL-6 and TNF-αexcept treatment group 5,compared with model group,the difference has statistically significant(P<0.05).Conclusions:Berberine can inhibite the proliferation and migration of diabetes mellitus rats VSMCs,can make the concentration of TGF-β1,IL-6 and TNF-αlower.